PROJECT SUMMARY The major obstacle to an 'HIV cure' is the persistence of viral reservoirs (VR) harboring replication competent viral genomes that have the capacity to produce infectious virus. These VR persist for long periods of time, and even after years of suppressive ART, the systemic spread of virus resumes within a few weeks upon cessation of ART in all but exceptional cases. Effective cure strategies will need to dramatically reduce or eliminate VR through safe and scalable approaches. It is currently thought that the major VR are long-lived latently infected resting memory CD4+ T cells, which remain quiescent until they are stimulated by external cues to produce virus. In addition to the truly latent VR, emerging data shows that in individuals on suppressive ART a subset of VR transcribe viral RNA (vRNA+) at variable levels (termed ?active VR?). In some cases, this might lead to residual levels of HIV replication, particularly in tissue microenvironments where drug concentrations are suboptimal. Even without full viral replication, this residual expression of virus may have adverse consequences and contribute to chronic immune activation/inflammation and non-AIDS defining clinical events. Eradicating HIV will require targeting both the ?latent? and ?active? VR, however, our current understanding of HIV reservoirs comes mostly from studies performed in peripheral blood, but the blood contains only a small fraction of VR during ART. We reason that to maximize efficacy of ?HIV cure? strategies, we need to first better characterize both the tissue compartments and the cellular subsets from which infection might rebound in HIV-infected individuals after ART is interrupted. Thus, the overarching goals of this research proposal, in response to RFA-AI-18-053 ?Single-Cell Multi-Omics of HIV Persistence?, is to merge our innovative in situ hybridization (ISH) approaches to quantify and map VR at high resolution with multiple new cutting-edge multi-omics platforms to investigate mechanisms of VR persistence at the single-cell level while retaining critically important contextual insight into the cellular immune neighborhoods and inflammatory landscapes in which VR reside. In Aim 1, we will utilize our suite of novel next-generation ISH (RNAscope, DNAscope and BASEscope) platforms to quantify and generate ?atlases? of ?latent? and ?active? VR longitudinally within tissue compartments (peripheral and mesenteric lymph nodes, spleen, GI tract) before and at different timepoints during ART anti-inflammatory adjunctive therapy. In Aim 2, we will perform an in-depth phenotypic analysis of VR and the cellular immune neighborhoods and inflammatory landscapes in which they reside within tissues (guided by our high-resolution in situ VR mapping outlined above) using Multiplexed Ion Beam Imaging (MIBI) proteomic analysis as well as unbiased SNaPP and nanoPOTs mass spectrometry approaches for spatiotemporal molecular analyses on samples obtained by LCM of immune neighborhoods and single cells, as well as on dissociated FACS sorted single cells. In Aim 3, we will perform in depth FISSEQ that combines the spatial context of RNA-FISH and the global transcriptome profiling of RNA-seq on tissue sections (as outlined above) but retained at the single-cell level.